Listeria monocytogenes isolates (n = 81) recovered from ready-to-eat meat-based food products (RTEMP) collected in industrial processing plants and retail establishments were genetically characterized for comparison with those from human clinical cases of listeriosis (n = 49). The aim was to assess RTEMP as a possible food source for human infection. L. monocytogenes was detected in 12.5% of the RTEMP samples, and in some cases, counts were above the European food safety criteria. All isolates were assessed by multiplex PCR for serogroup determination and detection of virulence-associated genes inlA, inlB, inlC, inlJ, plcA, hlyA, actA, and iap. Serogroups IIb and IVb dominated in RTEMP and human isolates, and all were positive for the assessed virulence genes. Antibiotic susceptibility testing by the disk diffusion method revealed a low level of resistance among the isolates. Pulsed-field gel electrophoresis (PFGE) of L. monocytogenes isolates, using restriction enzymes ApaI and AscI, revealed genetic variability and differentiated the isolates in five clusters. Although some pulsed-field gel electrophoresis profiles of particular RTEMP and human isolates seemed to be highly related, exhibiting more than 90% similarity, which suggests a possible common source, in most cases the strains were not genetically or temporally matched. The close genetic relatedness of RTEMP and human listeriosis strains stressed the importance of preventive measure implementation throughout the food chain.

The genus Listeria encompasses several species, but only Listeria monocytogenes and Listeria ivanovii are known to be human pathogens (10, 58). L. monocytogenes can lead to listeriosis, a severe illness with high fatality rates and long-term sequelae, which is almost entirely transmitted through the ingestion of contaminated foods (10, 35). Ninety percent of listeriosis cases occur in high-risk groups that include persons older than 65 years of age and immunocompromised individuals with bacteremia and central nervous system infections, as well as pregnant women and their neonates with preterm delivery, miscarriage, or stillbirth as a result (10, 19, 39). Healthy adults may suffer a febrile gastroenteritis after ingesting a large number of L. monocytogenes bacteria (39). Recently, a changing pattern of the disease has been observed, with an increasing number of cases among the elderly (31, 33, 53).

Currently, human listeriosis reference treatments combine aminopenicillin (ampicillin or amoxicillin) and gentamicin, while trimethoprim is generally used in case of intolerance to beta-lactams (1, 10). L. monocytogenes is widely susceptible to clinically relevant classes of antibiotics, although in vitro resistance was observed for older quinolones, fosfomycin, and expanded-spectrum cephalosporins (44).

L. monocytogenes strains can be classified into four major phylogenetic groups, even though the majority of L. monocytogenes isolates cluster in two lineages, named lineage I and II (12). Lineage I includes predominantly serogroups IIb and IVb but also IIc (respectively, related to serotypes 1/2b and 3b; 4b; and 3c), which are mostly adapted to the human host and capable of causing listeriosis. Lineage II strains include serogroups IIa and IIc (associated with serotypes 1/2a and 3a; and 1/2c, respectively), commonly found in foods, widespread in natural environments, and generally isolated from animal listeriosis and sporadic human clinical cases. Lineages III and IV strains, largely represent strains of serogroup IVa (serotypes 4a and 4c), are rare, and predominantly isolated from animal sources (45). Different L. monocytogenes serogroups diverge in their pathogenicity or in their ability to transmit to humans or both, and more than 90% of human listeriosis is linked to serogroups IIa, IIb, and IVb (35, 43).

The most common food categories implicated in listeriosis are ready-to-eat meat-based food products (RTEMP), soft cheeses, and smoked fish and shellfish (6, 15, 53, 54). RTEMP are of particular concern because they are able to support the growth of L. monocytogenes and can become contaminated through cross-contamination from the processing environment after the listericidal treatment step. Because RTEMP do not require a heat treatment prior to consumption, the pathogen may thrive (19, 57). L. monocytogenes can establish environment niches in food processing plants and retail establishments, where it is introduced through raw materials of different sources, or by personnel, because of its ubiquitous nature and widespread occurrence in the environment (51, 58). The RTEMP food chain needs to implement preventive measures regarding control and reduction of L. monocytogenes in final products, anticipating safety margins for temperature abuse and the possibility of a longer storage time (36). Also, the lack of knowledge by consumers on the importance of proper food storage and preparation might account for the increase of initial low levels of L. monocytogenes in RTEMP products.

The extended period of listeriosis incubation and the space-time–scattered distribution of cases hinder epidemiological surveillance and food source tracking and attribution, emphasizing the importance of highly discriminatory typing methods to reveal potential sources and routes of food contamination and human infection, which will further assist in the design of preventive strategies for disease control. This study aimed to establish a possible match of L. monocytogenes isolates from RTEMP collected in the producing industry and retail establishments with those from human sporadic cases of listeriosis, to assess RTEMP as a possible food source of human infection.

Food samples collection. One hundred twenty RTEMP samples were collected in 10 industrial processing facilities and nine retail establishments located in the central region of Portugal, particularly in the metropolitan region of Lisbon, between 2011 and 2013 (Table 1). In brief, in each industrial unit, two packaged RTEMP final products were collected, while the RTEMP samples collected in retail establishments were prepacked in the original package or sliced and packed by order at the delicatessen section. The criteria for RTEMP sample selection consisted of having the main ingredient as pork, veal, or poultry meat (or a combination of them) and going through a technological step that included cooking, baking, fermentation, drying, or smoking. These RTEMP samples were classified as able to support the growth of L. monocytogenes and not specially intended for infants or for special medical purposes, because they were handled after the thermal treatment, in operations, such as slicing, shredding, cutting, or packaging. However, some of the studied RTEMP samples had a fermentation, drying, or smoking step and due to their pH and water activity could be considered as not able to support the growth of L. monocytogenes (Table 1).

TABLE 1.

Characterization of all the assessed RTEMP samples distributed by food chain sampling point and year of collection

Characterization of all the assessed RTEMP samples distributed by food chain sampling point and year of collection
Characterization of all the assessed RTEMP samples distributed by food chain sampling point and year of collection
TABLE 1.

Continued

Continued
Continued

For practical purposes, industrial RTEMP samples were coded with the letter F, followed by the industry number, and another letter (A or B) representing the order of sample collection. Retail RTEMP samples were coded with the letter F, followed by pbo (packed by order) or pp (prepacked), and an additional number distinguishing sample collection order (Table 1).

Food samples were transported to the laboratory in an isothermal box (below 5°C) in less than 2 h and prepared (27). Detection and enumeration of L. monocytogenes was performed. Briefly, for L. monocytogenes detection, 25 g of each sample was homogenized in half Fraser broth (Scharlau, Barcelona, Spain) and incubated at 30°C for 24 h. Afterwards, 0.1 ml was transferred to 10 ml of Fraser broth and incubated at 37°C for 24 h. From each of the cultured Fraser broths, 0.1 ml was streaked onto agar Listeria according to Ottaviani and Agosti (ALOA; bioMérieux, Inc., Marcy l'Étoile, France) and incubated at 37 °C for 48 h. Whenever possible, up to 10 typical colonies were streaked from ALOA onto Trypticase soya agar (Scharlau), and plates were incubated at 37°C for 24 h (26). Confirmation of presumptive colonies was done by PCR (50, 52).

For enumeration of L. monocytogenes, 25 g of each sample was homogenized in half Fraser broth, and the suspension was spread onto ALOA plates that were incubated at 37°C for 24 or 48 h and presumptive typical colonies were enumerated (28). Confirmation of presumptive L. monocytogenes colonies was done by PCR (50, 52).

Human clinical isolates collection. L. monocytogenes isolates (n = 49) were provided by the Laboratory of Clinical Microbiology from Centro Hospitalar de Lisboa Norte in Lisbon, Portugal. All isolates were collected from 49 patients, 19 to 89 years old and from both sexes, from 2007 to 2013. L. monocytogenes was isolated from blood, cerebrospinal fluid, pus, bone, ascitic fluid, and amniotic fluid of infected individuals.

L. monocytogenes identification. L. monocytogenes identification was confirmed by PCR (Table 2) (50, 52). All isolates from food samples and human cases (n = 130) were assessed for serogroup determination and virulence factor gene detection.

TABLE 2.

Identity and nucleotide sequences of primer sets and PCR conditions used in this study

Identity and nucleotide sequences of primer sets and PCR conditions used in this study
Identity and nucleotide sequences of primer sets and PCR conditions used in this study

Virulence characterization. Serogrouping of all L. monocytogenes isolates (n =130) was done by multiplex PCR followed by an additional PCR for the flaA gene amplification (Table 2) (30). The identification of L. monocytogenes virulence genes was done by PCR amplification of the inlA, inlB, inlC, and inlJ genes (34), which code for internalin proteins A, B, C, J, respectively, and of plcA, hlyA, actA, and iap genes (Table 2) (47). Because all the isolates from the same food sample belonged to the same serogroup and presented the same virulence genes profile, one isolate up to, whenever possible, three isolates per food sample were used in the antibiotic susceptibility testing and further genetic characterization.

Antibiotic susceptibility testing. Antibiotic susceptibility testing of L. monocytogenes isolates (a total of 88 isolates of which 49 were from human clinical cases and 39 collected from RTEMP samples) was performed by the disk diffusion method on Mueller-Hinton agar (Scharlau) incubated at 37°C for 24 h (13). Disks containing commonly used antibiotics (Oxoid, Basingstoke, UK) in human and veterinary therapy were used: ampicillin (2 μg), amoxicillin-clavulanate (30 μg), ciprofloxacin (5 μg), erythromycin (15 μg), gentamicin (10 μg), linezolid (10 μg), meropenem (10 μg), benzylpenicillin (1U), quinupristin-dalfopristin (15 μg), rifampin (5 μg), sulphamethoxazole-trimethoprim (25 μg), tetracycline (30 μg), and vancomycin (5 μg). For quality control purposes, reference strain Staphylococcus aureus ATCC 25923 was used. For result interpretation, guidelines for L. monocytogenes were used (14), and for those antibiotic breakpoints not referred for L. monocytogenes, guidelines for gram-positive bacteria were used (8, 20).

PFGE typing. Genetic characterization of the isolates (n = 88) was performed by using the Centers for Disease Control and Prevention PulseNet standard procedure for L. monocytogenes typing (22). Bacterial genomic DNA in 1% agarose (SeaKem Gold Agarose, Cambrex Corporation, East Rutherford, NJ) plugs was digested in separate reactions with 10 U/μl of AscI (New England Biolabs, Ipswich, MA) for 4 h at 37°C, and with 50 U/μl of ApaI (New England Biolabs) for 4 h at 25°C, respectively. Pulse-field gel electrophoresis (PFGE) of the resulting DNA fragments was performed in 1% SeaKem Gold Agarose gels, with lambda PFG ladder standard (New England Biolabs) in 0.5× Tris-borate-EDTA buffer (NZYTech, Lisbon, Portugal) at 14°C, with 6 V/cm, initial pulsed time of 4.0 s and final pulsed time of 40 s, included angle of 120° over 19 h using a CHEF-DR III system apparatus (Bio-Rad Laboratories, Hercules, CA). Gels were stained with ethidium bromide (Sigma, St. Louis, MO) and photographed under UV transillumination.

Dendrograms were constructed based on L. monocytogenes PFGE patterns in BioNumerics software package Version 6.10 (Applied Maths, Sint-Martens-Latem, Belgium). L. monocytogenes PFGE patterns were analyzed to determine strain relatedness with an optimization setting of 1.5% and a band-position tolerance of 1.5% for AscI and ApaI restriction. Cluster analysis was performed by using the unweighted pair group method with arithmetic averages and band-based Dice correlation coefficient.

L. monocytogenes in RTEMP samples. L. monocytogenes was detected in 15 (12.5%) of 120 of the analyzed RTEMP samples, specifically in 5 of the 20 industrial samples and in 10 of the 100 retail samples. L. monocytogenes frequency (25%) in the industrial segment of the RTEMP chain is higher than the ones reported in the cooked and smoked pork sausages processing industry (42), in which 1.8% of the analyzed RTEMP samples were found to be positive for L. monocytogenes. Also, only 2% of positive samples were reported in smoked ham industrial processing facilities (46), while 8% of positive samples were found in fermented sausages processing plants (40). However, the high frequency of L. monocytogenes found in the present work in RTEMP samples collected from the producing industry is in line with other studies in Portugal. Twenty-five percent of ham samples collected from producers and retailers were found to be contaminated by L. monocytogenes (41). From our results, the high frequency of L. monocytogenes in industrial RTEMP seems to be due to specific nonconforming prerequirements related to selection and control of raw materials, equipment preventive maintenance, and the hygiene program (25).

L. monocytogenes frequency (10%) in the retail segment of the RTEMP chain is in line with the ones reported in similar studies at retail level, in which the frequency ranged from 5 to 20.5% (7, 9) and might be related with the long shelf life of these products, with the food handling practices and the hygiene at retail level (32). The positive samples were made of pork (70%), chicken (20%), and turkey (10%), which is in accordance with the proportion reported by European official authorities regarding L. monocytogenes presence in RTEMP samples (15). A collection of 81 L. monocytogenes isolates was obtained from industrial and retail RTEMP samples.

Only those samples that were sliced and packed by order in the retail delicatessen counter (Fpbo39, Fpbo46, Fpbo47, Fpbo48, and Fpbo49) and Fpp72 revealed counts ranging from 2.0 to 3.6 log CFU/g. These figures are above the European Union food safety criteria of 2.0 log CFU/g for ready-to-eat foods placed on the market during their shelf life, so these RTEMP samples are considered unsatisfactory. These counts above the food safety criteria limit might be due to inappropriate RTEMP handling and poor hygiene of the food preparation equipment, particularly the slicer, which was repeatedly used without any cleaning and sanitizing operations between RTEMP slicing, as observed during sampling. L. monocytogenes high counts are also associated with temperature failures and nonconforming sanitizing procedures (21, 25). Moreover, the original source of contamination might have been another RTEMP that was sliced before the collection of these samples or a food handler. In a study assessing L. monocytogenes in food processing plants, the bacteria was detected in 38% of the food handlers and more than one L. monocytogenes genotype was found on work surfaces, machines, and final products within the same processing plant (48).

L. monocytogenes human clinical isolates. The majority of L. monocytogenes isolates were obtained from affected individuals older than 60 years of age, regardless of sex (49% were men, while 51% were women). The human clinical cases collection addressed in our study reflects the changing pattern of human listeriosis in developed countries with well-established public health surveillance systems, in which reports reveal that listeriosis is affecting people older than 65 years more frequently than pregnant women, not only because of a higher life expectancy in those countries, but also because those individuals suffer from underlying disease(s) and are immunocompromised (31, 38).

Additionally, a seasonal pattern was observed with most of the cases occurring during spring and summer (60%), whereas in autumn and winter, only 40% were reported. This is in accordance with the large summer peaks and smaller winter peaks reported in Europe (15). Blood culture was the most common form of diagnosing L. monocytogenes infection in humans. However, no data regarding consumed foods, symptoms, disease evolution, and fatality were available, due to the lack of mandatory listeriosis notification in Portugal in the considered time frame (2007 to 2013).

L. monocytogenes serogroup assessment. Considering the serogroup distribution of all the positive L. monocytogenes RTEMP samples, the most frequent serogroup was IIb (33%), followed by IVb (27%), IIa (27%), IVa (7%), and IIc (7%). Serogroup IIb was found to be the most frequent in RTEMP collected in Nanjing, People's Republic of China, and previous works on L. monocytogenes serotype distribution in RTEMP in Europe indicate serotypes 4b (included in serogroup IVb) and 1/2a (included in serogroup IIa) as the most frequently reported (3, 4, 11, 24, 56).

The abovementioned packed by order RTEMP samples that presented counts above 2.0 log CFU/g belonged to serogroup IIb (Fpbo46, Fpbo47, Fpbo48, and Fpbo49) and serogroup IVb (Fpbo39 and Fpp72). Strains belonging to these serogroups exhibit an increased pathogenic potential to humans (12). Thus, these RTEMP samples with high counts of L. monocytogenes belonging to those serogroups more commonly related to human listeriosis present an increased risk for consumers.

Considering human clinical isolates (Table 3), serogroup IVb was the most frequent (67%), followed by IIb (18%) and IIa (14%). These serogroups are responsible for more than 90% of human listeriosis (43, 45, 51). A similar distribution of serogroups was found in Portugal among human isolates collected between 1994 and 2007 (2). Overall, these figures coincide with the ones reported by the European Food Safety Authority in the European summary report on trends and sources of zoonoses, zoonotic agents, and foodborne outbreaks in 2014 (15).

TABLE 3.

Human listeriosis isolates description and assessed serogroups

Human listeriosis isolates description and assessed serogroups
Human listeriosis isolates description and assessed serogroups

Virulence gene characterization. All RTEMP and human clinical L. monocytogenes isolates presented the virulence markers inlA, inlB, inlC, inlJ, plcA, actA, hlyA, and iap genes. However, those isolates belonging to serogroups IVa (n =10) and IVb (n =47) were not positive for the inlB gene. This fact might indicate the inability of the primers used to recognize the inlB gene of serogroups IVa and IVb and might be explained by the diversity presented by inlB gene in different L. monocytogenes serotypes (34). In fact, serotypes 4a, 4b, 4c, 4d, and 4e might present an altered inlB gene in relation to serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, and 3c (34).

The presence or absence of the assessed virulence genes might foretell the pathogenicity potential of the studied isolates, but because all the RTEMP and human clinical isolates presented the same virulence profile, this does not allow the differentiation of the isolates' virulence. This depends on the presence and activity of certain codons that might contribute to the development of the virulence mechanism in L. monocytogenes, which is still not fully understood (23).

Isolate antimicrobial testing. The frequency of antibiotic resistance on the tested L. monocytogenes isolates (n = 88) is very low (3%). All isolates were susceptible to the assessed antibiotics. However, the isolates (n = 3) from the industrial RTEMP sample F9B revealed a multidrug resistance profile to gentamicin, meropenem, benzylpenicillin, quinupristin-dalfopristin, rifampin, sulphamethoxazole-trimethoprim, and tetracycline. Note that these isolates belonged to serogroup IVa, which is rarely identified in food samples and human clinical cases of listeriosis (55), so the potential to induce human disease is low. Even so, resistance to gentamicin and trimethoprim is worrisome, because the former is usually the first-choice treatment coupled with ampicillin or amoxicillin, and trimethoprim is used in betalactam–intolerant patients (44). L. monocytogenes is acquiring resistance to a broad range of antibiotics, among which are those traditionally used to treat listeriosis, such as penicillin and gentamicin, and although not common, multiresistant strains are emerging (37). In L. monocytogenes isolates, 11.7% of resistance to at least one antibiotic was found in food and food-related environments, and resistance to one antibiotic was more common than multiple resistance (8). The antimicrobial resistance of L. monocytogenes isolates from RTEMP that were susceptible to first-choice antibiotics was assessed (57), and 100% of resistance to trimethoprim-sulphamethoxazole was exhibited. The observed antibiotic resistance in this study might be related to raw materials from animal origin that can be potential vehicles of antibiotic-resistant isolates to the food chain. The misuse of antimicrobials in food animals may trigger selective pressure for resistant L. monocytogenes isolates with posterior horizontal dissemination of antibiotic resistance genes (37). A resistance development mechanism could also be induced by exposure to sublethal doses of antimicrobial substances (additives) intentionally added to food and also to recurring exposure to sanitizers in food-related environments (1).

PFGE typing. All isolates (n = 88) were characterized by PFGE, and from an initially resulting dendrogram (not shown), it was possible to notice that isolates from the same RTEMP sample exhibited more than 95% similarity. For that reason, only one isolate per RTEMP sample was selected to be analyzed, together with all human isolates resulting in the dendrogram presented in Figure 1.

FIGURE 1.

Dendrogram of PFGE profiles from isolates in this study. In the sampling point column, numbers (industrial plants) and letters (retail establishments) refer to the food unit coding, and HSM is the acronym for Santa Maria Hospital.

FIGURE 1.

Dendrogram of PFGE profiles from isolates in this study. In the sampling point column, numbers (industrial plants) and letters (retail establishments) refer to the food unit coding, and HSM is the acronym for Santa Maria Hospital.

Close modal

Cluster A (Fig. 1) exhibits a mix of 12 RTEMP and human isolates, mostly belonging to serogroup IIa. High similarity is observed in the PFGE profile of human isolates in this cluster, as H28 and H38, although both belong to temporally distant clinical cases, indicating a possible common and persistent source. In contrast, considering the RTEMP strains in this cluster, different sources could have highly related PFGE profiles, as Fpp97, Fpp100, and Fpp35, which might be explained by the ubiquitous nature of L. monocytogenes (17, 49). Prepacked sliced ham (Fpp97) and prepacked shredded ham (Fpp100) were collected in the same retail establishment and according to its labels, produced in the same industry, and the corresponding isolates revealed 100% similarity with the same serogroup (IIa). This may highlight a possible common contamination source within the producing industry, because these samples were not handled in the retail establishment, as they were prepacked RTEMP. Additionally, strain Fpp35 presents a highly similar PFGE type (96%) with Fpp97 and Fpp100, despite being collected from a different retail establishment and produced in a different plant. In cluster A, none of the RTEMP and human isolates are related, with F2B and H11 being the most similar with 78% homology.

In cluster B (Fig. 1), PFGE profiles of L. monocytogenes isolates from RTEMP samples Fpbo46, Fpbo47, Fpbo48, and Fpbo49 are highly related (>97% similarity). Although these are four different RTEMP samples, they were prepared sequentially in the same slicing machine of the retail delicatessen; hence, a common source of contamination could be identified for these samples. Furthermore, the PFGE profiles of RTEMP isolates Fpbo46, Fpbo47, Fpbo48, and Fpbo49 share more than 88% homology with human isolates H32758 and H14667 that, in turn, seem to be clones (98% similarity) and also belong to the same serogroup (IIb). These human strains were collected 8 months apart from each other, from two clinical listeriosis cases in 2013 (Table 3), and although they are not directly linked to these particular RTEMP samples collected in 2011, their highly related PFGE profiles might point to a common source. Additionally, this finding may also be consistent with the occurrence of stable clonal groups of L. monocytogenes that might be present in foods and in food-related environments (17). According to Fugett et al. (18), molecular subtyping data have shown that L. monocytogenes can persist in processing environments for a considerable time (more than 10 years). Human contamination of foods and of the working environment cannot be discarded, because fecal carriage of Listeria occurs in 1 to 15% of the population (29).

In cluster C, human isolates H12 and H15 are clones isolated 8 months apart from each other in 2008 and 2009, sharing more than 91% similarity with RTEMP isolate F3A from an industrially processed roasted piglet collected in 2011. Also, human isolates H2, H24, and H37 (serogroup IIb) exhibit more than 86% homology with the above-mentioned strains. All these human clinical isolates seem to be involved in an invasive form of listeriosis and were isolated over four subsequent years (from 2007 to 2010). Again, this is highly suggestive of a stable strain persistence over time, in which a common source might be involved (16, 17); because the roasted piglet was cut in pieces after the listericidal treatment, cross-contamination could have occurred from the producing environment or from human sources. Also, in cluster C, PFGE profiles of RTEMP isolates Fpbo39 and Fpp13 are associated with human clinical isolates H31 (from 2010) and H36 and H40 (both from 2008) profiles, displaying more than 85% similarity and all belonging to serogroup IVb. In these cases, the possibility of a common source should not be discarded, but further studies, such as multilocus sequence typing analysis, must be considered (17). In fact, the upstream food chain continuum should be addressed in a root cause analysis to understand the origin and persistence of a common strain, including the animal husbandry farm, slaughterhouse, and food producing industry, where L. monocytogenes might persist in refrigerated environments for years (5). Nevertheless, based on the results, RTEMP could be potential food vehicles for human listeriosis infection, but other foods might also be involved, such as produce, seafood, and dairy (4, 11).

Cluster D is constituted exclusively by human isolates of serogroup IVb, relating only distantly with the RTEMP isolates in our work. Human isolates H29, H33, H34, and H41, identified in 2009, exhibit more than 87% of PFGE profile homology, suggesting temporally related cases of listeriosis. Also, isolates H45141 and H81683 are clones, even though they were isolated with a gap of 1 year. The genetic profiles of isolates H6, H7, H8, H9, and H10 exhibit 100% similarity and share more than 86% similarity with isolates H3, H25, and H27; these isolates were collected sequentially during 1 year and displayed a highly similar genetic profile, consistent with a common persistent food source, as listeriosis is mostly transmitted by ingestion of contaminated foods (10). Additionally, most of these isolates were collected from blood samples of individuals aged 60 years and older, which is in accordance with the changing pattern of human listeriosis that is currently affecting the elderly population more often (31).

In cluster E, PFGE profiles of human isolates H19 and H20 and RTEMP isolate F4B share 85% homology. These human isolates were collected with a temporal gap of 4 months, but RTEMP isolate F4B was collected 3 years after the clinical isolation, so although not matching temporally, their PFGE profiles display high similarity. Also, isolates H22 and H23 exhibit more than 93% homology, and both are related to RTEMP isolate Fpp72 with 85% similarity; still none are temporally connected. In both cases, the persistence of a stable strain might have occurred, and a possible common source could be involved.

The present study characterized L. monocytogenes strains from RTEMP and human clinical cases in an overlapping time in Portugal, during which no mandatory notification for human listeriosis was in place. For this reason, epidemiological data were scarce, considering affected patient information and consumed foods. Besides this limitation, the use of molecular subtyping provided important initial information about L. monocytogenes isolates in RTEMP and human clinical cases, allowing for their comparison.

From our data, however, it was possible to observe the persistence of strains over time and the possibility of common sources and routes of infection, emphasizing the need for preventive measure improvement along the RTEMP food chain continuum. Most importantly, to eliminate eventual environmental niches of L. monocytogenes, the following should be carefully considered and planned: the strict selection and control of raw material suppliers, enhancement of food handlers health status control, workers' training conducive to proper behaviors and attitudes toward food preparation, consistent hygiene procedures with adequate equipment sanitizing frequency, and programmed maintenance operations.

L. monocytogenes was detected in 12.5% of the RTEMP samples, and in some cases, counts were above the European food safety criteria. The majority of the isolates were found to be of serogroups IIb and IVb, and all presented the same virulence genes profile, whether in RTEMP samples or in human clinical samples, making them indistinguishable. Nevertheless, PFGE typing revealed genetic diversity of L. monocytogenes isolates that were gathered in five different clusters, with some particular RTEMP isolates sharing the same pulsotype or presenting high similarity with clinical isolates. This might point toward a common source related to cross-contamination from the food-producing environment, personnel involved in food processing operations or raw materials. However, in most cases, human and RTEMP strains did not match genetically or temporally.

Our work reinforces the need to address all the RTEMP food chain stakeholders when designing and implementing preventive and control measures for L. monocytogenes. All the operations after the listericidal treatment in RTEMP processing and handling should be carefully considered, particularly in retail establishments, to reduce the potential risk that these foods might represent to the consumer in the transmission of foodborne-acquired listeriosis.

The authors gratefully acknowledge the technical support provided by Maria Helena Fernandes, Maria José Fernandes, and Maria Paula Silva. This work was supported by the project “Portuguese Traditional Meat Products: Strategies To Improve Safety and Quality” (PTDC/AGR-ALI/119075/2010) and by Programa de Desenvolvimento Rural PA 13017 from Ministério da Agricultura, do Mar, do Ambiente e do Ordenamento do Território–Instituto de Financiamento da Agricultura e Pescas, I. P. The authors gratefully acknowledge the logistic support of Centro de Investigação Interdisciplinar em Sanidade Animal financed by Project UID/CVT/00276/2013 and the financial provision given by Fundação para a Ciência e Tecnologia with Ph.D. research grant SFRH/BD/70711/2010.

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